To find lead compounds for drug discovery programs, large numbers of compounds are often screened for their activity as enzyme inhibitors or receptor agonists/antagonists. Large libraries of compounds are needed for such screening. As a result of developments in this field, it is now possible to simultaneously produce combinatorial libraries containing hundreds of thousands of small molecules for screening. With the availability such libraries, however, has come a need for large scale, rapid screening methods.
For example, the libraries may be contained on microbeads, each compound being present in a picomolar amount. Because the amount of compound is very small, it is advantageous to conduct the high throughput screening method in very small volumes, e.g., on the order of 1 .mu.l. Such assays can be performed in the 1536 well plate described in U.S. patent application Ser. No. 60/037,636 filed Feb. 18, 1997. Microassays in such small volumes, however, are difficult to accurately and repeatedly perform using conventional methods.
Receptor binding assays used in high throughput screening typically involve three steps. First, a labelled ligand is incubated with a target receptor in the presence of compound to be tested for inhibition of ligand/receptor binding. Second, the receptor and ligand (and compound) are separated using filtration and/or washing of an immobilized receptor. Finally, the amount of labelled ligand bound to the receptor is quantified. This conventional screening is a `separations-mode` assay, i.e., one in which the bound ligand is physically separated from the free ligand using either a filtration membrane or the selective adhesion of either bound or free component to a surface (e.g., the surface of a microtiter plate).
Separation, however, is time-consuming and therefore slows high throughput screening. It can also, if fluid handling steps employed are not sufficiently precise, create variations in the signal generated in the assay and can disturb equilibrium binding conditions. Furthermore, separation is difficult to automate and is potentially hazardous when radioactive materials are involved. These problems are particularly acute in assays conducted in microvolumes using small amounts of test compound.
It is therefore advantageous in high throughput screening to distinguish bound and free ligands in a homogeneous assay, i.e., one that eliminates the need for separation. To be particularly useful in screening large scale combinatorial libraries, such an assay should readily permit small volumes, and small amounts of test compounds, to be used.
A homogeneous assay is described in U.S. Pat. No. 4,568,649 which employs beads that are impregnated with a scintillant (these are commercially sold as Scintillation Proximity Assay beads (SPA.TM., Amersham Corp., Arlington Heights, Ill.)). The beads are also coated with a ligand that is capable of binding with radio-labelled target in a sample. When the ligand binds to the radio-labelled target, the scintillant on the bead is activated by the radiolabel. The level of light energy produced by the scintillant indicates the amount of bound labelled target in the sample. This method, however, requires handling of radioactive reagents and is somewhat limited in sensitivity.
Another homogeneous assay is known in which signal is generated when labeled ligand and labeled target interact. One label is an energy donating Eu-cryptate having a long-lived fluorescent excited state and the other is an energy-accepting protein, allophycocyanin, having a short fluorescent excited state. Energy transfer occurs between the labels when they are less than 7 nm apart. During the assay, the Eu-cryptate is excited by a pulsed laser, and its fluorescent emission continually re-excites the allophycocyanin, whose fluorescence is measured by a time resolved fluorescence reader. This method, however, requires labeling of both the ligand and the target and is not as sensitive as some other commercially available assays. Also, allophycocyanin is a very large, multimeric protein which can affect the assay in an unpredictable manner.
A fluorescent imaging plate reader has been used to perform optical screening in cell-based kinetic assays that measure membrane potential and intracellular calcium. The assay employs an optical method that limits the depth of field measured by a CCD camera to the bottom of an assay well, where fluorescence in a layer of live adherent cells is measured. By limiting the depth of field to the cell layer, background fluorescence from extracellular dye is reduced. Data is obtained over time measuring, e.g., depolarization of cells (Schroeder et al., (1996) Journal of Biomolecular Screening 1:75-80). This method, however, uses live cells that require maintenance for the period of the assay, necessitating complicated integrated fluid handling to trigger rapid cellular events. Such handling is very difficult, if not impossible, to perform in the microvolumes that are used in high throughput screening of small amounts of library compounds. Also, measurement is taken of a bulk sample, i.e., of the entire layer of cells and the assay does not discriminate between fluorescence bound to individual suspended cells and background fluorescence.
It is therefore an object of the present invention to provide an assay for high throughput screening that does not require a separation step, i.e., is homogeneous.
It is another object of the invention to avoid radioactive waste, and to avoid labeling of both ligand and target molecule.
It is another object of the invention to provide an assay which is readily adaptable for miniaturization in microvolumes, and which is highly sensitive.
It is another object of the invention to provide a high throughput screening method for detecting activity of small amounts of compounds, such as are found in combinatorial libraries of beads having picomolar amounts of compound thereon.